Gut ORIGINAL ARTICLE Gut: first published as 10.1136/gutjnl-2015-309897 on 2 February 2016. Downloaded from Periodontitis induced by drives periodontal microbiota dysbiosis and insulin resistance via an impaired adaptive immune response Vincent Blasco-Baque,1,2,3,4 Lucile Garidou,1,2,3 Céline Pomié,1,2,3 Quentin Escoula,1,2,3 Pascale Loubieres,1,2,3,4 Sandrine Le Gall-David,5 Mathieu Lemaitre,4 Simon Nicolas,1,2,3 Pascale Klopp,1,2,3 Aurélie Waget,1,2,3 Vincent Azalbert,1,2,3 André Colom,1,2,3 Martine Bonnaure-Mallet,6 Philippe Kemoun,4 Matteo Serino,1,2,3 Rémy Burcelin1,2,3

►► Additional material is ABSTRACT published online only. To view Objective To identify a causal mechanism responsible Significance of this study please visit the journal online (http://dx.doi.org/10.1136/ for the enhancement of insulin resistance and gutjnl-2015-309897). hyperglycaemia following periodontitis in mice fed a fat-enriched diet. What is already known on this subject? 1INSERM U1048, Toulouse, Design We set-up a unique animal model of ▸ Cross-sectional studies show a positive France 2 periodontitis in C57Bl/6 female mice by infecting the correlation between periodontitis and type 2 Institut des Maladies fi Métaboliques et periodontal tissue with speci c and alive pathogens like diabetes. Cardiovasculaires (I2MC), Porphyromonas gingivalis (Pg), Fusobacterium nucleatum ▸ A diabetogenic fat-enriched diet induces Toulouse, France and Prevotella intermedia. The mice were then fed with periodontitis via increased periodontal 3 Université Paul Sabatier (UPS), a diabetogenic/non-obesogenic fat-enriched diet for up pathogens and lipopolysaccharides Toulouse, France 4Faculté de Chirurgie-Dentaire to 3 months. Alveolar bone loss, periodontal microbiota (LPS)-signalling activation. de Toulouse, Technical platform dysbiosis and features of glucose metabolism were ▸ Bacterial members of dental plaque have been of Research in Odontology, quantified. Eventually, adoptive transfer of cervical found in the microbiota of atherosclerosis Toulouse Cedex 09, France (regional) and systemic immune cells was performed to 5 plaques in humans. EA 1254 Microbiologie Risques demonstrate the causal role of the cervical immune ▸ http://gut.bmj.com/ infectieux—2, Rennes Cedex, A dysbiosis induces metabolic France system. disease. 6CHU Rennes et EA 1254 Results Periodontitis induced a periodontal microbiota ▸ An adaptive immune system induces metabolic Microbiologie Risques dysbiosis without mainly affecting gut microbiota. The disease. infectieux—2, Rennes Cedex, disease concomitantly impacted on the regional and France fi systemic immune response impairing glucose What are the new ndings? ▸ metabolism. The transfer of cervical lymph-node cells A high-fat diet (HFD) increases periodontal Correspondence to microbiota diversity in mice, conversely to that on September 28, 2021 by guest. Protected copyright. Dr Vincent Blasco-Baque and from infected mice to naive recipients guarded against periodontitis-aggravated metabolic disease. A treatment reported for gut microbiota. Pr Remy Burcelin, INSERM ▸ UMR1048-I2MC Team 2 with inactivated Pg prior to the periodontal infection abundance in periodontal “Intestinal Risk Factors, induced specific antibodies against Pg and protected the microbiota is linked to insulin resistance and Diabetes and Dyslipidemia” mouse from periodontitis-induced dysmetabolism. Finally, positively correlated to alveolar bone loss. Building L4, 1st floor, Hospital ▸ Periodontitis induces antibodies against of Rangueil 1, Avenue Jean a 1-month subcutaneous chronic infusion of low rates of Poulhès, BP 84225, 31432, lipopolysaccharides from Pg mimicked the impact of Porphyromonas gingivalis (Pg), and the fi Toulouse Cedex 4, France; periodontitis on immune and metabolic parameters. decrease of these speci c antibodies was [email protected] and Conclusions We identified that insulin resistance in associated with the impaired glucose [email protected] the high-fat fed mouse is enhanced by pathogen- metabolism in HFD-fed mice. ▸ Received 4 May 2015 induced periodontitis. This is caused by an adaptive Pg-LPS was shown to be responsible for the Accepted 18 December 2015 immune response specifically directed against pathogens periodontitis-induced metabolic impairment Published Online First and associated with a periodontal dysbiosis. under an HFD. 2 February 2016 ▸ The modulation of the regional (cervical) adaptive immune system is causally responsible for periodontitis-induced insulin resistance. INTRODUCTION Type 2 diabetes (T2D) is now considered a pan- demic disease. The causal origin of this accelerat- the diabetic population is 60% while it ranges ing development is related to several interacting from 20% to 50% in the general population.23In To cite: Blasco-Baque V, factors such as sedentary lifestyle, excessive body patients with periodontal diseases the incidence of Garidou L, Pomié C, et al. Gut weight (BW), stress and bad feeding habits.1 pre-diabetes or undiagnosed T2D is increased by 2017;66:872–885. Markedly, the prevalence of periodontitis within 27–53%.45Of note, treating periodontal diseases

872 Blasco-Baque V, et al. Gut 2017;66:872–885. doi:10.1136/gutjnl-2015-309897 Gut microbiota

microbiota and glucose metabolism have been also investigated.

Significance of this study We report the causal role of regional adaptive immune system Gut: first published as 10.1136/gutjnl-2015-309897 on 2 February 2016. Downloaded from response in the worsening of insulin resistance induced by peri- odontitis and precisely the LPS from Pg. An impaired specific How might it impact on clinical practice in the immune response against Pg could be responsible for the foreseeable future? enhancement of the incidence and the gravity of T2D. ▸ Increased Porphyromonadaceae abundance in the periodontal microbiota of diabetic patients may represent a MATERIALS AND METHODS new prognostic marker for periodontitis-aggravated insulin Animals and experimental procedures C57Bl/6J wild-type (WT) (Charles River, L’Arbresle, France) resistance. fi ▸ Pg-LPS-based inhibitory therapy and targeted female mice were group-housed (six mice per cage) in a speci c directly against Porphyromonadaceae may be useful to pathogen-free controlled environment (inverted 12 h daylight prevent the deleterious effects of periodontitis on glucose cycle, light off at 10:00). Five-week-old mice were randomised into two groups: group 1 was colonised (Co) and group 2 homeostasis in diabetic patients. 9 ▸ An anti-inflammatory strategy directed against the regional served as control. For group 1, 1 mL of a mix of 10 colony- forming unit (CFU) of each periodontal pathogen such as Pg immune system could reduce the incidence of insulin fi 22 resistance and type 2 diabetes. ATCC 33277, Fn and Pi previously identi ed, in 2% carboxy- ▸ A vaccination strategy against Pg may reduce the impact of methylcellulose was applied at the surface of the mandibular periodontitis on glucose metabolism. molar teeth, four times a week, during 1 month. Control mice received the vehicle only. Each group was divided into two sub- groups and fed with either a normal chow (NC, energy content: 12% fat, 28% protein and 60% carbohydrate; A04, reduced by 0.4% glycosylated haemoglobin in patients with Villemoisson-sur-Orge, France) or a diabetogenic, high-fat T2D.5 Nonetheless, the causal link between periodontitis and carbohydrate-free diet (HFD; energy content: 72% fat (corn oil T2D is still unknown. The last decade demonstrated that the and lard), 28% protein and <1% carbohydrate; SAFE, Augy, incidence of metabolic and cardiovascular diseases6 was also France) for 3 months.23 The groups were labelled as following: linked to gut microbiota dysbiosis.78Therefore, we reasoned NC+vehicle (NC), NC+colonisation (NC-Co), HFD and HFD that a dysbiosis of periodontal microbiota could be responsible, +colonisation (HFD-Co). at least in part, for incidence of metabolic diseases,9 and that the lipopolysaccharides (LPS) from Gram-negative Periodontal and gut microbiota analysis could be released in local and systemic organs, leading to meta- Total periodontal DNA was extracted from frozen mandibles bolic endotoxemia and insulin resistance as described in mice10 and faeces as previously described.24 For periodontal tissue, the and humans.11 whole 16S bacterial DNA V2 region was targeted by the Numerous Gram-negative LPS-releasing periodontal patho- 28F-519R primers and pyrosequenced by the 454 FLX Roche

gens are located within the periodontal biofilm. As an example, technologies at Research&Testing Laboratory (http://www. http://gut.bmj.com/ Prevotella intermedia (Pi) induces periodontal tissue destruc- researchandtesting.com/, Texas, USA). An average of 4907 tion12 and its prevalence is higher in patients with diabetes.13 sequences was generated per sample. For gut microbiota, the Interestingly, periodontal bacteria, such as Fusobacterium MiSeq technique was applied to generate an average of 10 000 nucleatum (Fn), have been found within the atherosclerotic sequences per sample by Research&Testing Laboratory. The plaque, suggesting a potential translocation towards the systemic cladogram in figure 1K and the linear discriminant analysis circulation.14 Moreover, Porphyromonas gingivalis (Pg) is asso- (LDA) score analysis in online supplementary figure S1 have ciated with chronic diseases such as cardiovascular diseases,15 been generated via the Galaxy website of Huttenhower’s Lab.25 on September 28, 2021 by guest. Protected copyright. rheumatoid arthritis,16 pancreatic cancer17 and non-alcoholic steatohepatitis (NASH).18 In fact, Pg is present in the injured Quantification of mandibular alveolar bone resorption liver and aggravates NASH via promoting inflammation.18 It has Hemi-mandibles were scanned using a high-resolution mCT been established that metabolic diseases are characterised by a (Viva CT40; Scanco Medical, Bassersdorf, Switzerland).26 Data chronic low-grade inflammation named metabolic inflamma- were acquired at 45 keV, with a 10 μm isotropic voxel size. Six tion,19 where macrophages and T-lymphocytes are recruited linear measurements were obtained from each molar by using a within metabolic tissues such as liver and adipose depots and stereomicroscope with an on-screen computer-aided measure- release proinflammatory cytokines such as tumour necrosis ment package. The alveolar bone loss (ABL) (mm) was measured factor (TNF)-α, interleukin (IL)-1β, IL-6 and plasminogen acti- from the cemento-enamel junction to the alveolar bone crest for vator inhibitor (PAI)-11 that impair insulin action. Hence, each molar.27 Three-dimensional reconstructions were generated insulin resistance seems to be secondary to the onset of an from a set of 400 slices. inflammatory process,1 in which innate and adaptive immune responses may promote inflammatory reactions driven by gut Real-time quantitative PCR analysis for periodontal tissue microbiota.20 21 Total RNA from periodontal tissue was extracted using the Altogether, these evidences suggest that a periodontal micro- TriPure reagent (Roche, Basel, Switzerland). cDNA was synthe- biota dysbiosis could initiate first a regional and then a systemic sised using a reverse transcriptase (Applied Biosystems, Fost metabolic inflammation promoting insulin resistance and T2D. City, USA) from 1 mg of total RNA as previously explored.28 To demonstrate the causal role of periodontal diseases as a risk The primers (Eurogentec, San Diego, USA) used were (50 to 30): factor for T2D and the relevance of the innate and adaptive TNF-α, forward TGGGACAGTGACCTGGACTGT; reverse, immune responses, we have set-up a unique and specific model TCGGAAAGCCCATTTGAGT; IL-1β, forward TCGCTCA of periodontitis by Gram-negative bacterial periodontal- GGGTCACAAGAAA; reverse CATCAGAGGCAAGGAGG pathogen colonisation in mice. Features of periodontal AAAAC; PAI-1, forward ACAGCCTTTGTCATCTCAGCC;

Blasco-Baque V, et al. Gut 2017;66:872–885. doi:10.1136/gutjnl-2015-309897 873 Gut microbiota Gut: first published as 10.1136/gutjnl-2015-309897 on 2 February 2016. Downloaded from http://gut.bmj.com/ Figure 1 Oral colonisation with Porphyromonas gingivalis (Pg), Fusobacterium nucleatum (Fn) and Prevotella intermedia (Pi) induces periodontitis associated with local and systemic immune disorders. (A) Mice were colonised with Pg, Fn and Pi or by vehicle solution for 1 month and then randomised into four groups: normal chow (NC, blue bar, n=6), normal chow colonised (NC-Co, purple bar, n=6), high-fat diet (HFD red bar, n=7) and high-fat diet colonised (HFD-Co, green bar, n=10). (B) Hemi-mandible for each group, as reconstructed by micro-CT (Viva CT40; Scanco Medical, Bassersdorf, Switzerland). (C) Alveolar bone loss (yellow line) for each group. (D) Tumour necrosis factor (TNF)-α, plasminogen activator inhibitor (PAI)-1, interleukin (IL)1-β and IL-6 expression in periodontal tissue. (E) Histological examination for hemi-mandibles (PT, periodontal tissue; T, tooth) stained with H&E, F4/80, CD3 and CD20 antibodies: cells count is shown on the right side of each panel series. Arrows show infiltrated cells within the anatomical area and insets show a magnification of the slide. Number and relative abundance of immune cell types explored at 3 months in on September 28, 2021 by guest. Protected copyright. cervical lymph nodes (F and G) and in spleen (H and I) and the blood (J). (K) Linear discriminant analysis effect size (LEfSe) analysis-based cladogram for periodontal microbiota of each group. (L) Complete linkage clustering using Euclidean distance. Data (mean±SEM) and one-way analysis of variance (ANOVA) followed by Tukey’s test used for *p<0.05 and ****p<0.0001 when compared to HFD, §p<0.05; §§p<0.001 §§§§p<0.0001 when compared to NC and $p<0.05 when compared to NC-Co. reverse CCGAACCACAAAGAGAAAGGA; and IL-6 forward described.10 After full recovery from the surgery and 6 h ACAAGTCGGAGGCTTAATTACACAT; reverse TTGCCATTG of fasting, the whole-body glucose utilisation rate was CACAACTCTTTTC. The concentration of each mRNA was evaluated in euglycemic/hyperinsulinemic conditions, as normalised for RNA loading against the ribosomal protein L19 described elsewhere.10 (RPL19) (forward GAAGGTCAAAGGGAATGTGTTCA; reverse CCTTGTCTGCCTTCAGCTTGT) as an internal stand- Histological analyses −ΔΔCT ard, and the data were analysed according to the 2 Hemi-mandibles were excised, fixed in 4% paraformaldehyde 28 method. for 48 h and embedded in paraffin. Hemi-mandibles samples were cut with a microtome in the transverse direction following Intraperitoneal glucose-tolerance test and in vivo the main axis of tooth from coronal to apical. Then, sections euglycemic/hyperinsulinemic clamp (4 mm thickness) were stained with H&E. Immuno-histological Six-hour fasted mice were injected with glucose into the peri- analyses were performed using primary antibodies against F4/80 toneal cavity (1 g/kg). Blood glucose was measured with a gluc- (AbD Serotec, Colmar, France), CD3 (Spring Bioscience, ometer (Roche Diagnostics, Meylan, France) on 2 μL of blood Pleasanton USA) and CD20 (Thermo Scientific, Rockford, collected from the tip of the tail vein at −30, 0, 30, 60 and USA), and revealed by R.T.U. (Ready-to-Use) Vectastin Elite 90 min after the glucose injection. To assess insulin sensitivity, a (Vector Laboratories, Burlingame, USA) and for diaminobenzi- catheter was indwelled into the femoral vein as previously dine (DAB) by ImmPACT DAB Substrate (Vector Laboratories),

874 Blasco-Baque V, et al. Gut 2017;66:872–885. doi:10.1136/gutjnl-2015-309897 Gut microbiota Gut: first published as 10.1136/gutjnl-2015-309897 on 2 February 2016. Downloaded from http://gut.bmj.com/ on September 28, 2021 by guest. Protected copyright.

Figure 1 Continued

Blasco-Baque V, et al. Gut 2017;66:872–885. doi:10.1136/gutjnl-2015-309897 875 Gut microbiota Gut: first published as 10.1136/gutjnl-2015-309897 on 2 February 2016. Downloaded from http://gut.bmj.com/ on September 28, 2021 by guest. Protected copyright.

Figure 1 Continued

876 Blasco-Baque V, et al. Gut 2017;66:872–885. doi:10.1136/gutjnl-2015-309897 Gut microbiota

to quantify the infiltration of immune cells. Slides were scanned plasma was separated and frozen at −80°C. Also, 10 mLof

with ‘panoramic digital scanner 250’ with Z-Stack function and plasma were used to determine insulin concentration with an Gut: first published as 10.1136/gutjnl-2015-309897 on 2 February 2016. Downloaded from the objective 40× (3DHISTECH). Cells subpopulations count- Elisa kit (Mercodia, Uppsala, Sweden) following the manufac- ing was done with the Panoramic Viewer software turer’s instructions. Plasma cytokine concentration was deter- (3DHISTECH) and carried into the Lamina propria gingivae mined by the MILLIPLEX MAP system (Luminex, Austin and periodontal ligament on average surface 0.1 mm2 on each 12212 Technology Blvd., Austin, Texas, USA/Merck Millipore tissue section. Five microscopic fields were counted on each Headquarters 290 Concord Road Billerica, Massachusetts, slide by two independent naïve investigators. USA).

Surface staining and antibodies treatment of immune cells Statistical analysis from cervical lymph nodes, spleen and blood Results are presented as mean±SEM. One-way analysis of vari- Mononuclear cell suspensions were incubated for 15 min with ance (ANOVA) followed by Tukey’s post-test was used to assess anti-CD16/32 to block Fc receptors and then with antibodies, inter-groups differences, except for the IPGTT, where two-way anti-CD4 APC (RMA4-5, eBioscience), CD8 V450 (53.6.7, BD ANOVA followed by Bonferroni’s post-test was applied. Bioscience), anti-CD11b APC-eFluor780 (M1/70, eBioscience), *p<0.05; **p<0.01; ***p<0.001 and ****p<0.0001 when CD45 V500 (30F11, BD Bioscience), anti-CD19 FITC (1D3, compared to HFD, §p<0.05; §§p<0.001 §§§§p<0.0001 when BD Bioscience) and anti-TCR PerCP-Cy5.5 (H57, eBioscience) compared to NC and $p<0.05 when compared to NC-Co for 30 min on ice. LIVE/DEAD Fixable Cell Stain Kit (Life defined statistical significance. Statistical analyses were per- Technologies) was used to remove dead cells. All data were formed using GraphPad Prism V.5.00 for Windows Vista acquired using a digital flow cytometer (LSR II Fortessa, Becton (GraphPad Software, San Diego, California, USA). The dendro- Dickinson), and analysed with FlowJo software (Tree Star). gram on figure 1L was drawn by PermutMatrix software;30 figure 2I (principal coordinate analysis (PCoA)) was drawn by Immunotherapy XLSTAT for Microsoft Windows Excel. Cervical lymph nodes were harvested either from mice colo- nised with bacteria mixture as described above or not colonised. RESULTS Cervical lymph node cells (107 total) were injected into the Pg, Fn and Pi periodontal pathogens promote periodontal, peritoneal cavity from mice with periodontitis (periodontitis cervical and systemic immune disorders together with transfer cells) or without (healthy transfer cells) and an intraper- periodontal microbiota dysbiosis during an HFD itoneal glucose-tolerance test (IPGTT) was assessed after trans- Pg, Fn and Pi,31 periodontal pathogens, are drivers of the fer and after colonisation with periodontal pathogens. development of periodontitis in mice. Here, to demonstrate that periodontitis is an aggravating risk factor for diet-induced meta- Immunisation bolic disease, we generated a unique mouse model. First, peri- An injection of 106 CFU of Pg, Fn or Pi or the mix of the three odontitis was induced by colonising 5-week-old WT C57Bl6/J bacteria, inactivated by oxygen-exposure during 48 h, was given female mice with all three pathogens; then, mice were fed with

in the footpad muscle. Control mice were injected with saline. an NC or a diabetogenic/not obesogenic HFD (figure 1A). http://gut.bmj.com/ Then, periodontitis was induced (as described above) 1 month We validated our model by showing periodontal after the immunisation in 3 months HFD-fed mice. pathogens-induced mandibular ABL, a feature of periodontitis, on NC. Moreover, this parameter was worsened on HFD Pg-LPS treatment (figure 1B, C). Then, we studied the periodontal tissue looking Mice were subcutaneously implanted with an osmotic mini- for a putative inflammatory status. As shown in figure 1D, pump (Alzet Model 2004; Alza, Palo Alto, California, USA) NC-fed colonised mice displayed a significant increased gene delivering either ultrapure LPS from Pg at 300mg/kg/day as pre- expression for all the analysed cytokines. Moreover, the on September 28, 2021 by guest. Protected copyright. viously reported10 (CAYLA-InvivoGen, Toulouse, France) or pro-inflammatory effect due to TNF-α and PAI-1 was increased NaCl (0.9%) to ensure a continuous 28 days infusion. Then, by HFD (figure 1D). Subsequently, given this evidence, we ana- mice were fed a NC or HFD for 1 month. Finally, mice were lysed histological sections of hemi-mandibles. We showed by sacrificed by cervical dislocation and tissues were collected and H&E staining that cells infiltrated the periodontal tissue snap-frozen in liquid nitrogen. (figure1E) after the colonisation with the periodontal pathogens on NC. In addition, we characterised the cell types by immunos- Anti-Pg antibodies measurement taining and showed an increased macrophage (cells F4/80+), Immunoglobulin G antibodies specific to LPS of Pg were mea- lymphocyte T (cells CD3+) and lymphocyte B (cells CD20+) sured using a home-made ELISA. The wells of 96-well flat- number in the same experimental conditions. In response to the bottom microtiter plates were coated in triplicates with LPS of HFD treatment, the number of cells increased when compared Pg. After washing and blocking the plates, serum samples were to NC-fed mice. Additionally, in colonised HFD-fed mice the added to individual wells and specific mouse IgG antibodies number of immune cells even further increased over that of NC, were detected with an alkaline phosphatase-conjugated anti- NC-Co and HFD mice, showing the impact of the dietary treat- mouse immunoglobulin. The absorbance was read at 405 nm ment and of the colonisation on the inflammatory process in using an ELISA plate reader. The results were expressed as an periodontal tissue (figure 1E). ELISA index (EI), which was the mean OD 405 of a given Next, to identify whether periodontitis and local inflamma- serum sample divided by the mean OD 405 of the calibrator tion may be associated with an impaired immune system, we (reference serum).29 quantified local (cervical lymph node) and systemic (spleen) adaptive and innate immune system cells. HFD feeding Plasma biochemical assays increased the number of cells in both cervical lymph node and Fifty microlitres of blood were sampled from the retro-orbital spleen when compared to NC-fed mice. Interestingly, periodon- sinus in awake condition in 6- h-fasted mice. For insulin, the titis blunted this increase in HFD-fed mice only (figure 1F, H).

Blasco-Baque V, et al. Gut 2017;66:872–885. doi:10.1136/gutjnl-2015-309897 877 Gut microbiota Gut: first published as 10.1136/gutjnl-2015-309897 on 2 February 2016. Downloaded from http://gut.bmj.com/ on September 28, 2021 by guest. Protected copyright.

Figure 2 Periodontitis enhances high-fat diet (HFD)-induced metabolic disorders in mice. Glycaemic profiles (mg/dL) during an intraperitoneal glucose-tolerance test (IpGTT; normal chow (NC, blue bar, n=6), normal chow colonised (NC-Co, purple bar, n=6), high-fat diet (HFD, red bar n=7) and high-fat diet colonised (HFD-Co, green bar, n=10)) and glycaemic indexes as inset; ratio fat/lean for each group during 1 month (A and B), 2 months (C and D) and 3 months (E and F). (G) Insulin sensitivity evaluated by the euglycemic-hyperinsulinemic clamp technique. (H) Correlation between glucose infusion rate (GIR) and alveolar bone loss (ABL). (I) Principal coordinate analysis (PCoA) between dominant bacterial families from periodontal microbiota (abundance >1%, detected at least in one mouse) and metabolic parameters such as ABL, GIR, gingival inflammation (TNFaG, IL1bG, PAI1G, IL6G, where ‘G’ stands for gingival), immunoglobulin G score, glycaemic index, fasted glycaemia and body weight at 3 months (IgG3, GI3, FG3, BW3, respectively): the three insets represent the correlation between GIR and Lactobacillaceae family, Porphyromonadaceae family and Porphyromonadaceae family with ABL. Data are mean±SEM. Significant results when *p<0.05; **p<0.01 and ***p<0.001 when compared to HFD, §p<0.05 and §§§p<0.001 when compared to NC and $p<0.05 when compared to NC-Co as determined by two-way analysis of variance (ANOVA) with Bonferroni’s post-test for (A), (C) and (E) and one-way ANOVA followed by Tukey’s post-test for (B), (D), (F) and (G).

In the latter, this variation was due to a strong reduction in the periodontitis increased the frequency of T-lymphocyte and frequency of innate CD11b+ cells (gating on CD3-CD19- B-lymphocyte (CD4+, CD8+ and CD19+) during HFD only CD11c-CD11b+) in cervical lymph nodes and spleen, whereas (figure 1G, I).

878 Blasco-Baque V, et al. Gut 2017;66:872–885. doi:10.1136/gutjnl-2015-309897 Gut microbiota Gut: first published as 10.1136/gutjnl-2015-309897 on 2 February 2016. Downloaded from

Figure 2 Continued

To further explore the systemic effect of periodontitis, we S2A). In HFD-fed mice, we also observed tiny modifications of analysed immune cells in blood, where the above reported mod- gut microbiota due to colonisation (only unclassified bacteria), ifications were confirmed for all cell types and especially for whereas HFD increased the intestinal proportion of dendritic cells (CD11b+CD11c+) and innate CD11b+CD11c− Proteobacteria (see online supplementary figure S2B). All the cells (figure 1J). By contrast, periodontitis had no significant gut microbial profiles in each group from Phyla to are impact on any cell type whatever the tissue under NC (figure provided in online supplementary figure S2C–H.

1F–J). Indeed, the periodontal colonisation increased the Altogether, this data set provides evidence that periodontal http://gut.bmj.com/ number of antibodies anti-Pg on NC, whereas the HFD treat- pathogens induce periodontitis in mice, alter periodontal micro- ment reduced IgG serum levels and antibodies anti-Pg in colo- biota and that the subsequent periodontitis aggravates nised mice only (table 1). Conversely, HFD increased IgG serum HFD-induced periodontal and systemic inflammation. levels independently of colonisation at both 2 and 3 months of treatment (table 1). Moreover, the periodontitis increased blood Periodontitis increases HFD-induced metabolic diseases IL-6 on HFD and decreased blood interferon-γ concentrations To demonstrate that periodontal pathogen-induced periodontitis on HFD and NC at 3 months (table 1). may represent an aggravating risk factor for diet-induced meta- on September 28, 2021 by guest. Protected copyright. Then, to evaluate whether the change in immune features was bolic diseases, we characterised glucose metabolism in response linked to a periodontal microbiota dysbiosis, we studied the to the nutritional stress. Our data show that periodontitis aggra- composition of periodontal microbiota on both NC-fed and vated the HFD-induced glucose intolerance by the first and up HFD-fed colonised mice. On NC, colonisation significantly to the third month of treatment (figure 2A, C, E). These data increased the genus Lactococcus, as shown by the cladogram were associated with a progressive and significant increase in the based on the LDA effect size (figure 1K). On HFD, colonisation fat/lean mass ratio (figure 2B, D, F). Furthermore, according to significantly increased the Porphyromonadaceae family, in the time course, periodontitis increased plasma leptin and accordance with Pg colonisation (figure 1K). We also showed insulin concentrations on HFD at 3 months (table 1). Insulin that HFD induces a periodontal microbiota dysbiosis based on resistance (indexed by the glucose-infusion rate (GIR)), as the significant increase in genera Bacteroides, Clostridium and assessed by the euglycemic/hyperinsulinemic clamp technique, Ruminococcus. The LDA scores and the details (Phyla to was induced by the periodontitis in HFD-fed mice only (figure Species) for all the periodontal microbiota changes are reported 2G). Importantly, ABL was strongly and significantly correlated in online supplementary figure S1. Interestingly, periodontitis (R2=0.52; p<0.0001) with insulin resistance (figure 2H). affected the overall periodontal microbiota profile of HFD-fed Then, we analysed by PCoA whether correlations may exist mice to a greater extent than NC-fed mice, as reported by between families abundance in periodontal microbiota and cluster analysis at the family level (figure 1L). metabolic parameters such as GIR, glycaemic index (GI3), We also investigated whether colonisation with periodontal fasted glucose (FG3), ABL, BW3, cytokines for gingival inflam- pathogens may induce changes in the gut microbiota. In NC-fed mation, blood immunoglobulins levels and gingival (IgG) cyto- mice, the above periodontal microbiota changes were associated kines at 3 months. We found that Lactobacillaceae abundance in with tiny modifications of gut microbiota due to colonisation, periodontal microbiota positively and significantly correlated which increased members of Actinobacteria and with GIR (figure 2I, up-left inset). By contrast, Deltaproteobacteria groups (see online supplementary figure Porphyromonadaceae abundance was negatively and significantly

Blasco-Baque V, et al. Gut 2017;66:872–885. doi:10.1136/gutjnl-2015-309897 879 Gut microbiota

correlated with GIR (figure 2I, up-right inset), but positively

with ABL (figure 2I, down-right inset). Overall, the PCoA Gut: first published as 10.1136/gutjnl-2015-309897 on 2 February 2016. Downloaded from allowed identifying a specific cluster for each group, despite a tiny superposition between the NC-Co and the HFD group. Furthermore, Porphyromonadaceae abundance was associated with gingival TNF-α expression (figure 2I, down-left inset), whereas we also found a positive and significant correlation between ABL and fasted glycaemia at 3 months (figure 2I, middle-right inset). Altogether, these data show that periodontitis aggravates HFD-induced glucose intolerance and insulin resistance together with changes in the periodontal microbiota, among which some of them are associated with the metabolic phenotypes. HFD-Co

Pg colonisation recruits cells from the adaptive immune

2±1 16±6* 18±5* 22±4* system to control glucose tolerance To demonstrate that the adaptive immune system was triggered by the change in periodontal and was a causal mechanism responsible for the deleterious impact of the peri- odontal pathogens on metabolic disease, we first transferred the cervical lymph-node cells from mice with or without periodon- titis to healthy recipient mice (figure 3A). In such conditions, the glucose tolerance was similar in both groups of recipient mice (not shown), suggesting that other factors were required to trigger the metabolic disease. Hence, we challenged the recipi- 2±1 4±1 ent mice with the periodontal pathogens during 4 weeks after HFD the cell transfer (figure 3A) and demonstrated that the glucose tolerance was improved in mice that received the immune cells from the infected mouse when compared to those that received

9±2 14±5 21±4immune 15±3 17±4 cells from 21±4 a non-infected 15±3 mouse (figure 3B, C). BW gain was not significantly affected (not shown). Then, to study the impact of the colonisation and of the transfer of cervical immune cells from mice with on

HFD-induced glucose intolerance, we then challenged the colo- http://gut.bmj.com/ nised and transferred mice for 4 weeks with HFD. After 4 weeks of HFD, the IPGTT is showing that the protection, although still significantly present in glycaemic index, is starting to vanish (figure 3D, E). The data show that when the HFD-fed mice were transferred with immune cells from mice with peri-

NC-Co odontitis, the glucose tolerance is improved although modestly (figure 3D, E). By contrast, the transfer of immune cells by on September 28, 2021 by guest. Protected copyright. itself, without colonising the recipient mice, was not sufficient to impact glucose tolerance (figure 3B–E). However, glucose metabolism could be impacted when the immune cells were spe- cifically adapted to the periodontal pathogens. This suggests that both nutritional stress and periodontitis are required factors to trigger metabolic phenotypes. In a second set of experiments, to further validate the role of the adaptive immune system on the control of glucose tolerance, we immunised the lymphocytes to the periodontal pathogens by treating mice with different sets of inactivated periodontal

4±1 8±2 9±4 19±8§ 21±10§ 8±5 fi 73±1722±722±6 108±17 26±10 138±25 28±5 22±8 100±33 46±22 16±1 87±13 24±4 95±10 13±4 23±5 81±18 33±12 93±12 25±11 100±19 32±9 96±15 30±8 110±18 126±7 20±3 30±9 27±8 36±25 230±106 255±420 13±8§ 7±5§ 14±7§pathogens 55±45 ( gure 176±80 4A). 70±30 The intramuscular 4±2* 7±3* treatment 23±30* with the 465±17741±505 440±11 683±376 483±32 760±385 472±18 940±263§ 626±503 462±33 384±153 518±18 1626±589§ 2023±451§ 636±36§ 2347±254§ 736±57§ 2209±63#* 784±109§ 1177±418#* 3474±421#* 671±41# 732±62# 836±105#* 1 month 2 months 3 months 1 month 2 months 3 months 1 month 2 months 3 months 1 month 2 months 3 months 1411±82 1199±73 2836±857 1468±91 1360±388 2231±561 1690±522 3199±1230§ 5933±947§ 991±98#* 2211±384#* 2362±514* NC three inactivated periodontal pathogens prevented the above s test used for *p<0.05 when compared to HFD, §p<0.05 when compared to NC and #p<0.05 when compared to NC-Co. ’ reported aggravating effects of periodontitis on HFD-induced

(EI) 1.45±0.24 1.54±0.24 1.49±0.14 3.89±2.44§glucose 3.76±2.63§ 3.58±1.82§ intolerance 1.19±0.11 1.12±0.25 at 1.92±1.70 1 month 1.79±0.45# 1.17±0.27# (see 1.31±0.29# online supplementary figure S3A–F), 2 months (not shown) and 3 months (figure 4B–F). Importantly, this preventive effect was due to Pg since the treat- ment of the mice with this unique bacterium was sufficient to P. gingivalis -

Systemic metabolic and inflammatory parameters in fasted mice over the time course of the treatment protect against periodontitis-induced metabolic diseases. Anti Moreover, the specific treatment by Pg prevented the decrease g/mL

, pg/mL of antibodies anti-Pg observed on HFD after periodontal colon- m γ fi N=6 per group; dataOne-way as ANOVA mean±SEM. followed by Tukey HFD, high-fat diet (n=6); HFD-Co, high-fat diet colonised (n=6) in the table; IFN, interferon; IL, interleukin; NC, normal chow (n=6); NC-Co, normal chow colonised (n=6). isation at 1 and 3 months (see online supplementary gures S3G IP10, pg/mL MIG, pg/mL RANTES, pg/mL Antibodies Parameters Insulinemia, pg/mL Leptinemia, pg/mL IgG, IL-6, pg/mL IFN- Table 1 and S4G). At 3 months of HFD, the treatment with Pg

880 Blasco-Baque V, et al. Gut 2017;66:872–885. doi:10.1136/gutjnl-2015-309897 Gut microbiota Gut: first published as 10.1136/gutjnl-2015-309897 on 2 February 2016. Downloaded from http://gut.bmj.com/ on September 28, 2021 by guest. Protected copyright.

Figure 3 Immune cells transfer from cervical lymph nodes from periodontitis mice reduce colonisation-induced glucose intolerance. (A) Immune cells from cervical lymph nodes from donor mice with or without periodontitis were transferred to recipient mice. Then, each group was colonised by Porphyromonas gingivalis (Pg), Fusobacterium nucleatum (Fn) and Prevotella intermedia (Pi) in periodontal tissue for 4 weeks. Intraperitoneal glucose-tolerance tests were performed in recipient mice after transfer (not shown), after colonisation (B) and after 4 weeks of high-fat diet (HFD) (D); (C) and (E) glycaemic index. After colonisation (±): HTC+NC-Co (black bar n=4): healthy transfer+colonisation, PTC+NC-Co (green bar n=4) periodontitis transfer+colonisation, HTC+NC (blue bar n=4) and PTC+NC (purple bar n=4) periodontitis transfer+no colonisation and after 4 weeks of HFD: HTC+HFD-Co (black bar n=4): healthy transfer+colonisation, PTC+HFD-Co (green bar n=4) periodontitis transfer+colonisation, HTC+HFD (blue bar n=4) and PTC+HFD (purple bar n=4) periodontitis transfer+no colonisation. Data are mean±SEM. Significant results when ***p<0.001 when compared to HTC+NC as determined by one-way analysis of variance (ANOVA) followed by Tukey’s test (C) and two-way ANOVA with Bonferroni’s post-test (B).

protected against the periodontal colonisation-induced ABL in infusion of a low rate of Pg-LPS aggravated HFD-induced periodontal tissue (figure 4H). glucose intolerance and insulin resistance (figure 5B–D). Furthermore, the fat to lean ratio was significantly increased in The LPS from Pg enhance HFD-induced metabolic diseases mice pre-treated with Pg-LPS when compared to saline In the quest for molecular determinants responsible for (vehicle)-pre-treated mice (figure 5E), suggesting that the LPS Pg-aggravated diet-induced metabolic diseases, we evaluated the from Pg could be a major molecular mechanism explaining the impact of LPS from Pg (figure 5A). A 1-month continuous metabolic impact of periodontitis on metabolic disease.

Blasco-Baque V, et al. Gut 2017;66:872–885. doi:10.1136/gutjnl-2015-309897 881 Gut microbiota Gut: first published as 10.1136/gutjnl-2015-309897 on 2 February 2016. Downloaded from

Figure 4 Pre-treatment with inactivated Porphyromonas gingivalis (Pg) prevents periodontitis-aggravated glucose intolerance in high-fat diet (HFD)-fed mice. (A) Mice were injected by 106 colony-forming unit (CFU) of inactivated Pg or inactivated Fusobacterium nucleatum (Fn)or inactivated Prevotella intermedia (Pi) or a mix of all inactivated bacteria or vehicle solution. One month later, mice were colonised by Pg, Fn,

Pi and/or by vehicle solution for 1 month and then randomised into seven groups: NC-Vehicles (vehicle+normal chow, black bar, n=4), HFD http://gut.bmj.com/ (vehicle+HFD, red bar, n=4), high-fat diet colonised (HFD-Co) (vehicle+HFD+colonisation, green bar, n=4), HFD-Co+I B mix (inactivated mix bacteria+colonisation+HFD, black blue bar, n=4), HFD-Co+I Pg (inactivated Pg+colonisation+HFD, purple bar, n=4), HFD-Co+I Fn (inactivated Fn+colonisation+HFD, light blue bar, n=4) and HFD-Co+I Pi (inactivated Pi+colonisation+HFD, orange bar, n=4). Intraperitoneal glucose-tolerance test (IpGTT) and glycaemic index were assessed for each group after 3 months of HFD (B–F); (G) measurement of immunoglobulin G antibodies specific to lipopolysaccharide (LPS) of Pg in blood. (H) Alveolar bone loss was explored after experimental procedures for each group. Data are mean ±SEM. Significant results when: **p<0.01, ***p<0.001 and ****p<0.0001 when compared to HFD vehicles, §p<0.05 and §§§§p<0.0001 when compared to NC vehicles and #p<0.05 and ####p<0.0001 when compared to HFD-Co as determined by two-way analysis of variance (ANOVA) with Bonferroni’s post-test for (B), (C), (D) and (E) and one-way ANOVA followed by Tukey’s post-test for (F) and (G). on September 28, 2021 by guest. Protected copyright.

DISCUSSION colonised mice. Conversely, periodontitis induced a profound Here, we report that periodontitis and notably the LPS from Pg periodontal microbiota dysbiosis, aggravated on HFD. are aggravating factors for HFD-induced glucose intolerance Among periodontal pathogens, Pg is a key candidate in the and insulin resistance. The underlying mechanism is associated relationship between periodontitis and general health.32 Pg has a with the capacity to produce specific antibodies against Pg. specific arsenal of virulence factors that enable it to invade the By using our unique animal model, we show that, in mice at periodontal tissue and subsequently disseminate into the systemic risk of developing metabolic diseases (ie, HFD-fed), the prior circulation. First, the ability of Pg to invade the periodontal colonisation of periodontal tissue with Pg enhances the develop- biofilm is regulated by specific enzymes such as the bacterial ment of glucose intolerance by affecting the adaptive immune dipeptidyl peptidase 4 (DPP4).33 As an indication, inhibitors of system response against Pg. This set of data confirms evidences DPP4 are currently used to treat T2D, suggesting that the effi- in humans where the incidence of metabolic diseases is further cacy of this therapeutic strategy may include the inhibition of raised when associated with periodontitis,23an epidemiological prokaryotic enzymes.34 Indeed, Pg is also capable of invading feature also observed in pre-diabetic or undiagnosed T2D host cells such as epithelial and endothelial cells.35 Pg is captured patients.45Our data show that the periodontitis itself was not by host cells through interactions between its fimbriae and sufficient to induce metabolic impairments since no change in α5β1-integrin expressed by the epithelial cells, allowing it to glycaemia or in insulin resistance was observed in infected cross the epithelial barrier.36 Altogether, these observations NC-fed mice. The corroborating factor brought by HFD could confer an important role of Pg on its dissemination. Here, we be linked to the gut microbiota dysbiosis characterised in show that part of Pg virulence specifically attributed to metabolic patients with impaired metabolism.14 However, periodontitis disease is through LPS since the continuous low rate infusion of appeared to induce no major changes in the gut microbiota of LPSs from Pg, together with an HFD, impacted on the immune

882 Blasco-Baque V, et al. Gut 2017;66:872–885. doi:10.1136/gutjnl-2015-309897 Gut microbiota Gut: first published as 10.1136/gutjnl-2015-309897 on 2 February 2016. Downloaded from http://gut.bmj.com/

Figure 5 Lipopolysaccharide (LPS) from Porphyromonas gingivalis (Pg) aggravates high-fat diet (HFD)-induced metabolic diseases. (A) Mice were

implanted with Pg-LPS or vehicle releasing minipumps for 1 month; then, mice were fed an HFD for 1 month. After 1 month of diet, on September 28, 2021 by guest. Protected copyright. glucose-tolerance (B), glycaemic index (C), insulin sensitivity (E) and ratio fat/lean mass (D) were assessed in each group: (Pg-LPS, red bar, n=7) or (NaCl, black bar, n=7). Significant results when: *p<0.05, **p<0.01 and ***p<0.001 when compared to HFD+vehicle as determined by one-way analysis of variance (ANOVA) followed by Tukey’s test, except for ‘B’ (two-way ANOVA and Bonferroni’s post-test).

system and consequently on insulin and glucose tolerance. By did not develop metabolic10 41 and periodontal22 diseases, which providing a mechanism, our conclusions thus confirm and reinforces our present conclusion. We cannot rule out that the further extend the data from the literature, which show that putative role of periodontitis on metabolic disease could be patients with periodontitis are characterised by the presence of linked to changes of gut microbiota. The sequencing data Pg-LPS into the blood.32 The continuous infusion of LPS from showed some subtle changes following the colonisation in Pg may induce a chronic low-grade systemic inflammation NC-fed mice. However, no impact on glucose nor on immune leading to the resetting of cardiac homeostasis.37 Such a mechan- homeostasis was detected in our experimental conditions. ism involves the toll-like receptors (TLRs) TLR-2 and TLR-438 Therefore, it could be expected that the periodontitis could leading to the activation of MyD88 and nuclear factor κB slowly impair metabolic features over a longer period of time (NF-κB) responsible for the transcription of pro-inflammatory even in the absence of nutritional stress. But we cannot rule out cytokines genes such as TNF-α or IL-1β.38 Furthermore, Pg that such mechanisms, as reported elsewhere,42 could have some LPS-stimulated gingival fibroblast showed NF-κB-dependent long-term impact in the present experimental conditions. The TNF-α production leading to an inflammatory process involving subtle changes in gut microbiota reported here have most likely ERK, p38 and JNK activation, as well as PAI-1 expression.39 no influence on glucose homeostasis. The mechanism through This mechanism was also observed in stem cells from human which Pg would favour the development of metabolic diseases periodontal ligament.40 The role of LPS to cellular cross-talk is could be due to the strong impact of microbiota dysbiosis on the essential to the virulence of Pg. We previously provided a mech- immune response.43 We show that following the action of Pg, a anism by showing that mice deprived of the LPS receptor CD14 pro-inflammatory process is triggered. The nutritional stress

Blasco-Baque V, et al. Gut 2017;66:872–885. doi:10.1136/gutjnl-2015-309897 883 Gut microbiota impacted the cervical and systemic immune response mostly Competing interests None declared. from the innate immune cells with a mild impact on Ethics approval All animal experimental procedures were approved by the local Gut: first published as 10.1136/gutjnl-2015-309897 on 2 February 2016. Downloaded from B-lymphocyte and T-lymphocyte. However, when combined ethical committee of Rangueil University Hospital (Toulouse, France). with the nutritional stress (HFD), this response was normalised, Provenance and peer review Not commissioned; externally peer reviewed. fl suggesting that the low-grade in ammation induced by the Open Access This is an Open Access article distributed in accordance with the innate immune system was under the control of the adaptive Creative Commons Attribution Non Commercial (CC BY-NC 4.0) license, which immune system. We demonstrated this hypothesis by showing permits others to distribute, remix, adapt, build upon this work non-commercially, that the transfer of cells from the cervical lymph nodes of and license their derivative works on different terms, provided the original work is infected mice to naive recipient mice prevented a further impact properly cited and the use is non-commercial. See: http://creativecommons.org/ licenses/by-nc/4.0/ of periodontitis on metabolic parameters. Therefore, the lym- phocytes from the colonised mice were educated to the peri- odontal antigens and protected the recipient mouse from the REFERENCES fl 1 Kolb H, Eizirik DL. Resistance to type 2 diabetes mellitus: a matter of hormesis? metabolic in ammation. Furthermore, we could notice the pro- Nat Rev Endocrinol 2012;8:183–92. duction of the antibodies anti-Pg in response to the treatment 2 Albandar JM, Rams TE. Global epidemiology of periodontal diseases: an overview. with the corresponding attenuated bacteria. 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